Novel Cosmetic Ingredient CS‐AA Polyion Complex and Skin Moisturizing Effect

ABSTRACT

Purpose

The study explored the enhanced skin moisturizing capabilities and moisture retention effects achieved by forming a polyion complex using sulfated glycosaminoglycan (GAG), specifically chondroitin sulfate (CS), and amino acids (AA) such as glutamine (Q) and arginine (R). The overall hydration effect of this CS‐AA complex was examined.

Methods

After analyzing the CS‐AA polyion complex structure using spectroscopic methods, the ex vivo moisture retention ability was assessed under dry conditions using porcine skin samples. Additionally, the efficacy of the CS‐AA polyion complex in reducing transepidermal water loss (TEWL) and improving skin hydration was evaluated on human subjects using a digital evaporimeter and a corneometer, respectively.

Results

Validating a systematic reduction in particle size, the following order was observed: CS > CS/AA simple mixture > CS‐AA complex based on dynamic light scattering (DLS) and transmission electron microscopy (TEM) analysis. Furthermore, observations revealed that the CS‐AA complex exhibits negligible surface charge. Additionally, Fourier‐transform infrared spectroscopy (FT‐IR) analysis demonstrated a distinct peak shift in the complex, confirming the successful formation of the CS‐AA complex. Subsequently, the water‐holding effect through porcine skin was assessed, revealing a notable improvement in moisture retention (weight loss) for the CS‐Q complex: 40.6% (1 h), 20.5% (2 h), and 18.7% (4 h) compared to glycerin. Similarly, the CS‐R complex demonstrated enhancements of 50.2% (1 h), 37.5% (2 h), and 33% (4 h) compared to glycerin. Furthermore, TEWL improvement efficacy on human skin demonstrated approximately 25% improvement for both the CS‐Q complex and CS‐R complex, surpassing the modest 12.5% and 18% improvements witnessed with water and glycerin applications, respectively. Finally, employing a corneometer, hydration changes in the skin were monitored over 4 weeks. Although CS alone exhibited nominal alterations, the CS‐Q complex and CS‐R complex showed a significant increase in moisture levels after 4 weeks of application.

Conclusion

In this study, polyion complexes were successfully formed between CS, a sulfated GAG, and AA. Comparisons with glycerin, a well‐known moisturizing agent, confirmed that the CS‐AA complex exhibits superior moisturizing effects in various aspects. These findings suggest that the CS‐AA complex is a more effective ingredient than CS or AA alone in terms of efficacy.

1. Introduction

Glycosaminoglycans (GAGs) are widely distributed in various tissues from different biological origins, particularly in the connective tissue of animals [1, 2, 3]. These linear polysaccharide chains are composed of repeating disaccharide units, with each disaccharide consisting of a hexosamine (N‐acetyl‐glucosamine or N‐acetyl‐galactosamine) and an uronic acid (L‐iduronic or D‐glucuronic acid) or galactose. The extent of sulfation varies depending on the GAG family [4, 5]. GAGs have been recognized as a valuable focus in skincare due to their significant presence in the skin, their ability to enhance skin hydration by restoring the skin barrier, and their decreased expression/activity in aging skin. Similar to the well‐known hyaluronic acid (HA), sulfated GAGs such as chondroitin sulfate (CS), dermatan sulfate (DS), heparin sulfate (HS), and keratan sulfate (KS) also exhibit strong biological activities that can be modulated to improve skin quality [6, 7, 8].

CS is an important carbohydrate found extensively in the skin and joints of both humans and animals. As a key component of the extracellular matrix, it has been shown to possess various beneficial biological effects [9, 10]. CS has been used as an injection for arthritis treatment and more recently, as a health functional supplement for arthritis [11, 12]. In terms of skin regulation, it effectively promotes the expression of fibroblast growth factors and reduces inflammatory factors. Additionally, CS significantly stimulates collagen synthesis, which is crucial for wound healing as collagen synthesis is a repetitive and time‐consuming process throughout the entire healing process [13, 14, 15]. Especially, CS presents great potential as a material for repairing a compromised skin barrier due to its abundant hydroxyl (–OH) groups and impressive water retention properties. Nevertheless, CS possesses a negative charge owing to sulfate groups, leading to its tendency to aggregate and making its absorption into the skin a difficult task [16, 17].

Amino acids (AAs) play a vital role in regulating skin hydration and pH to maintain skin health. They are major components of the skin's natural moisturizing factors and have been widely used in cosmetic skincare products, primarily for their moisturizing benefits [18, 19, 20, 21]. Arginine (R) is not only a component of the natural moisturizing factor but also a potent antioxidant that plays a crucial role in cell division, collagen synthesis, wound healing, and skin immunity [22, 23, 24]. Glutamine (Q), as the most abundant essential AA in the body, is important for protein synthesis and serves as an intracellular energy source. It has demonstrated various effects such as regulating cell proliferation, gene expression, anti‐inflammatory properties, immune function control, and antioxidant activity [25, 26]. Recent research has focused on using AA such as R and Q to reduce skin irritation, repair skin damage, and improve skin conditioning by forming AA‐based complexes [27, 28].

In cosmetic industry, there has been an extensive research and documentation on novel efficient ingredients. However, the actual number of efficient substances that can be practically utilized is limited due to concerns regarding stability and safety. Consequently, recent studies have been directed toward enhancing the efficacy by improving skin permeation of active ingredients and optimizing stability within the formulation, to address these issues [29, 30]. Among various techniques reported, the polyion complex approach, a type of ion pairing, stands out as a remarkable strategy. The polyion complex refers to the formation of complexes between a polymer and a substance with different surface charges, utilizing interactions such as charge–charge and hydrogen bonding. The resulting complexes exhibit reversible characteristics, as they do not involve actual bonding like surface modification or covalent bonding. It has been reported that the polyion complex method effectively reduces the influence of surface charges, thereby increasing the efficacy of transdermal delivery [31, 32]. Consequently, this technology holds significant appeal for enhancing the efficient absorption and efficacy of cosmetic active ingredients.

In this study, CS‐R and CS‐Q complexes were manufactured, and their structure was analyzed using techniques such as dynamic light scattering (DLS), transmission electron microscopy (TEM), and Fourier‐transform infrared spectroscopy (FT‐IR) to confirm the formation of polyion complexes. Finally, three moisturizing evaluations were performed to confirm the enhanced moisturizing effect through complex formation. First, water‐holding capacity in porcine skin was analyzed through ex vivo evaluation. Based on these results, a simple in vivo evaluation was conducted to observe the effect of reducing transepidermal water loss (TEWL) and evaluate the increase in water‐holding capacity. Finally, the overall skin hydration was assessed using a corneometer.

2. Materials and Methods

2.1. Formation of CS‐Q and CS‐R Complex

L‐Arginine (R, Ajinomoto), L‐glutamine (Q, Sigma–Aldrich), and sodium chondroitin sulfate (CS, Yantai Dongcheng Biochemicals) were prepared for the CS‐Q and CS‐R polyion complex in a molar ratio of 1:1 (CS:Q and CS:R, respectively). Deionized (DI) water was added until the final CS concentration reached 5%, and the complex solution was stirred at 50–70°C for approximately 20–30 min until a homogeneous and uniform yellowish liquid without precipitates was formed. CS/Q and CS/R simple mixture was prepared by dispersing them in DI water and mixing at room temperature until the final concentration of CS‐Q and CS‐R polyion complex matched. Each sample, including CS solution, CS‐Q and CS‐R polyion complex solutions, and CS/Q and CS/R simple mixture solutions, was treated with 2% 1,2‐hexanediol for preservation before completion of the samples. Before conducting in vitro and in vivo testing, the pH of the CS solution, CS‐Q, and CS‐R polyion complex solutions were adjusted to approximately 5.5–6, respectively, using a citric acid solution. No additional purification was performed.

2.2. Structure Analysis

To confirm the formation of the CS‐AA polyion complex, the following evaluations were conducted. First, the changes in size distribution (agglomeration) and zeta‐potential of CS, CS/Q, and CS/R simple mixtures, and CS‐Q and CS‐R complexes were examined. The size distribution and agglomeration of CS, CS/Q, and CS/R simple mixtures, and CS‐Q and CS‐R complexes were investigated by using DLS with a Zetasizer Nano ZS instrument (Malvern Instruments, UK). Scattered light was detected at an angle 173° using noninvasive back scattering (NIBS) technique. All solutions were measured at a standard material (CS) concentration of 0.1% to determine the change in particle size. Zeta‐potential measurement was carried out using a Zetasizer Nano ZS instrument (Malvern Instruments, UK). Triplicate samples were measured three times each at 25°C. The measurement was initiated within 120 s after sample preparation. Secondly, the trend in particle structure observed through Zetasizer analysis was confirmed by TEM images. TEM images for CS, CS/Q, and CS/R simple mixtures, and CS‐Q and CS‐R complexes were acquired using a JEM‐F200 instrument (JEOL, Japan). The coagulation and complex were dispersed on the copper grids, followed by pipetting a drop of solution that was left to evaporate overnight before analysis. Lastly, Fourier‐transform infrared spectroscopy (FT‐IR, Spectrum Two, Perkin Elmer) analysis revealed the occurrence of energy changes in the functional groups using attenuated total reflectance (ATR) mode due to the intermolecular interaction between CS, AA, CS/AA simple mixture, and CS‐AA complex. Through complementary interpretation of the data, the difference between the CS‐AA complex and the CS/AA simple mixture was elucidated, confirming the formation of the polyion complex.

2.3. Measurement of Water‐Holding Capacity Using Porcine Skin

A weight‐loss experiment was conducted to establish a dehydration model for predicting the water content in porcine skin [33, 34]. Porcine skin membranes with dimensions of 2.5 × 2.5 cm were purchased (Micropig Franz Cell Membrane, Apures, Korea). The mass of the skin samples (n = 4) was measured using a precision balance at 0, 1, 2, and 4 h intervals. Before the interval tests, the porcine skin samples were dried at room temperature. Each porcine skin sample underwent a simulated absorption process by applying an equal amount sample and rolling with a tip a consistent number of times. The weight immediately after sample absorption was measured and used as a reference point. Each sample was then exposed to drying conditions at approximately 35°C using a heating chamber. The samples were measured at each time point (0, 1, 2, and 4 h), which were subsequently normalized for comparison. It was expected that the lower the water‐holding capacity of the hygroscopic substance, the less the weight loss of the porcine skin due to dehydration. The normalized weight ratios were compared and analyzed for CS, DI‐water, glycerin at each time point, as well as between the CS/AA mixture and CS‐AA complex, taking into account significant differences.

2.4. Measurement of TEWL

TEWL was measured by using Tewameter TM300 (Courage and Khazaka electronic GmbH). Five sites were selected on the forearm of each of the seven volunteers (n = 7), and at each location, DI water, glycerin, CS solution, CS‐R complex, and CS‐Q complex were topically applied at an active substance concentration of 0.1%. The samples were consistently applied every day, using a consistent amount. Prior to each application, measurements were taken, and the values obtained 30 min and 1 week later were normalized to the corresponding pre‐application values. The TEWL reduction rates were then calculated and compared.

2.5. Measurement of Skin Hydration

Skin hydration was determined using the corneometer MPA580 (Courage and Khazaka Electronic GmbH). To assess the long‐term moisturizing effects of the CS‐AA complex on actual humans, a 4‐week evaluation was conducted at the same forearm spot. Seven participants were assessed throughout 4 weeks, with daily applications of a consistent amount at the same site. The assessment involved comparing the skin hydration before application and after the 4 weeks to determine (arbitrary units).

2.6. Statistical Analysis

All statistical analyses were performed using IBM SPSS Statistics 21.0 software (IBM, Armonk, NY). The results were expressed as the mean ± standard deviation (SD). Since we evaluated moisture levels in both synthetic and human skin samples, we normalized the values with respect to the pre‐application measurements for each sample and compared them. Differences between time points or groups were analyzed using t‐tests with the statistical significance set at p < 0.05.

3. Results

3.1. Preparation and Characterization of CS‐AA Complex

In order to investigate the formation of a specific structure through the binding of CS, R, Q, and their simple mixtures and complexes, the structure was analyzed using TEM microscopy. Each sample was prepared with a CS concentration of 0.5% and analyzed by TEM image as shown in Figure 1. Figure 1a represents the CS solution, while Figure 1b,c corresponds to the CS/Q simple mixture and CS‐Q complex, respectively. Similarly, Figure 1d,e represents the CS/R simple mixture and CS‐R complex. As shown in Figure 1a, the reported literature demonstrates that CS aggregates significantly when dispersed in water, as confirmed by TEM image. This aggregation is attributed to the polymer properties, leading to the formation of large aggregates without any specific orientation or structure. However, the CS/Q simple mixture and CS‐Q complex (Figure 1b,c) reduce aggregation phenomenon, even at the same concentration of 0.5% as the CS solution. Although, it is evident that some improvement in aggregation phenomena is observed through the decrease in aggregate size in the CS/Q simple mixture, particularly in the CS‐Q complex, noteworthy reduction in aggregation phenomena compared to CS and CS/Q simple mixture is confirmed. This can be attributed to the interaction between CS and Q through complex formation, which leads to a decrease in the self‐aggregation phenomenon, one of the specific characteristics of polymers.

FIGURE 1.

TEM image of (a) CS, (b) CS/Q simple mixture, (c) CS‐Q complex, (d) CS/R simple mixture, and (e) CS‐R complex. CS, chondroitin sulfate; Q, glutamine; R, arginine.

In the case where R is applied, similar to the CS/Q simple mixture, a decrease in aggregation phenomena is observed in both the CS/R simple mixture and the CS‐R complex. The CS/R simple mixture exhibits a reduction in the size of aggregates without the formation of specific structures. Conversely, the CS‐R complex does not show the formation of clear particulate structures like the CS‐Q complex, but rather exists in the form of smaller aggregates. Nonetheless, the CS‐R complex demonstrates the most significant improvement in reducing aggregation phenomena when R is applied, suggesting the formation of polyion complex, similar to the CS‐Q complex. These observed differences can be attributed to the characteristics or binding affinities between the amino acids Q and R employed in complex formation. Nevertheless, from a broader perspective, it can be concluded that the formation of polyion complexes through the binding of CS and AA reduces the self‐aggregation of CS and results in the formation of distinct structures differing from CS, taking into account the characteristics or interaction energy between Q and R employed in complex formation.

The mitigation of aggregation phenomena through the formation of CS/Q and CS/R simple mixtures, and CS‐Q and CS‐R complexes was validated by TEM images. To assess the formation of polyion complexes, the size and zeta‐potential of materials dispersed in water were investigated (Table 1). Initially, particle size analysis was conducted to validate the trends observed in the TEM images. In CS‐Q case, the particle size exhibited a descending order of CS, CS/Q simple mixture, and CS‐Q complex, as shown in Table 1. Significantly, the CS‐Q complex displayed a very small particle size, providing support for the assumption of specific structural formations. Additionally, the zeta‐potential, a crucial parameter for determining polyion complex formation, was examined. In the CS/Q simple mixture, both positive and negative zeta‐potential values were measured, indicating the preservation of Q's zwitterion characteristics. However, the zeta‐potential value was undetectable in the CS‐Q complex, indicating the successful formation of a polyion complex. Similarly, the size and zeta‐potential of CS‐R case were also assessed (Table 1). Although relatively larger than CS‐Q complex, the CS‐R complex exhibited a smaller size compared to the mixture, consistent with the trends observed in Figure 1d,e. Furthermore, when measuring the zeta potential, the mixture retained the zwitterion characteristics of R, with both positive and negative values. However, the zeta potential was not detectable in the CS‐R complex, confirming the formation of a polyion complex.

TABLE 1.

Size and zeta‐potential of CS, Q, CS/Q simple mixture, CS‐Q complex, R, CS/R simple mixture, and CS‐R complex.

	CS	Q	CS/Q simple mixture	CS‐Q complex	R	CS/R simple mixture	CS‐R complex
Size (nm)	5400	1.5	2500	100	1.25	930	620
Zeta‐potential (mV)	−18	33/−90	16.3/−21	—	−4	44.2/−13.6	—

Abbreviations: CS, chondroitin sulfate; Q, glutamine; R, arginine.

FT‐IR analysis was conducted to confirm the formation of polyion complexes by examining the energy changes between the functional groups. Polyion complexes formed by charge‐charge interactions or hydrogen bonding are influenced by the intermolecular interactions between the two substances, causing a shift in the position of the IR peaks. Figure 2a shows the FT‐IR spectra of the CS/R simple mixture and CS‐R complex, while Figure 2b shows the FT‐IR spectra of the CS/Q simple mixture and CS‐Q complex. In Figure 2a, the CS‐R complex exhibited a red shift at 2900 cm⁻¹ (O─H stretching of carboxylic acid) and 1602 cm⁻¹ (guanidinium group) compared to the mixture. Similarly, in Figure 2b, the CS‐Q complex showed a red shift at 2983 cm⁻¹ (O─H stretching of carboxylic acid), 1644 cm⁻¹ (C═O stretching), and 1520 cm⁻¹ (N─H stretching of the amino group) compared to the mixture. These results indicate that through the CS‐AA complex formation process, a stronger interaction occurred between the two substances through intermolecular interactions, distinguishing the complexes from simple mixtures. Together with the previous analysis results, it can be concluded that both CS‐R complex and CS‐Q complex formations resulted in intermolecular structure rather than CS‐AA simple mixtures.

FIGURE 2.

IR spectroscopic analysis of (a) CS/R simple mixture and CS‐R complex and (b) CS/Q simple mixture (black line) and CS‐Q complex (red line). CS, chondroitin sulfate; Q, glutamine; R, arginine. CS, chondroitin sulfate; Q, glutamine; R, arginine.

3.2. Evaluation of CS‐AA Complex in Water‐Holding Capacity, TEWL, and Skin Hydration

To investigate the enhanced moisturizing effect of CS through the formation of CS‐AA complex, various evaluations were conducted. First, a weight retention evaluation using porcine skin was performed in an ex vivo setting. The samples were dried at 35°C chamber and the weight was measured at 1, 2, and 4 h after absorption. The initial weight after absorption was used as the baseline for normalization and comparison. Glycerin, a well‐known humectant, was selected as the positive control group, with both CS and glycerin evaluated at an active concentration of 1%. Based on Figure 3, it can be observed that both water and glycerin 1% exhibited a similar trend of weight reduction ratio. However, it was found that CS 1% demonstrated a lesser weight loss ratio at each specific time interval. Therefore, under identical drying conditions, it can be inferred that CS possesses superior water‐holding capacity compared to glycerin and water. As shown in Figure 3, both CS/AA simple mixture and CS‐AA complex exhibited improved weight reduction compared to the sole use of CS. This observation suggests a synergy effect between CS and AA, leading to an enhancement in water‐holding capacity. The CS‐R complex showed an improved weight loss ratio compared to glycerin, with values of 50.2% (1 h), 37.5% (2 h), and 33% (4 h) at each respective time interval. Likewise, the CS‐Q complex demonstrated an improved weight loss ratio compared to glycerin, with values of 40.6% (1 h), 20.5% (2 h), and 18.7% (4 h). Although R is well‐known for its excellent moisturizing effect, making it challenging to attribute the synergy effect to both CS/R simple mixture and CS‐R complex, this is not the case for Q, which lacks such moisturizing properties. Therefore, it can be concluded that the water‐holding capacity was enhanced by the synergy effect between CS/Q simple mixture and CS‐Q complex. Furthermore, both the CS‐R complex and CS‐Q complex exhibited lesser weight reduction compared to their respective mixtures, indicating improved moisture retention. Consequently, these results highlight the reinforcing effect of polyion complex formation on the moisturizing effect. To evaluate the efficacy in reducing TEWL in human skin, we conducted TEWL analysis as part of our experimentation.

FIGURE 3.

Water‐holding capacity by changes in normalized weight of porcine skin interval time. Table data are represented as the mean ± SD, comparison between simple mixture and complex at each time and comparison between CS, DI water, and Glycerin at each time (*p < 0.05). CS, chondroitin sulfate; DI, deionized; SD, standard deviation.

Figure 4 demonstrates the changes in TEWL immediately 30 min after applying the samples and after 1 week (with daily application throughout the week), based on the TEWL values measured before applying each sample. It is evident that even application of DI water alone results in an approximate 10% decrease in TEWL after 30 min, increasing to around 12.5% after 1 week. The TEWL analysis also indicates that the CS solution, at an equivalent concentration, exhibits superior TEWL reduction effects both immediately after application and after 1 week, surpassing the efficacy of the widely used moisturizer, glycerin. These results align with previous assessments of weight retention in porcine skin (water‐holding capacity). Further analysis, including structural evaluation and skin moisture holding capacity, confirm that the CS‐AA complex demonstrates a synergistic effect that is at least comparable to that of the CS/AA simple mixture. Consequently, this evaluation excluded the CS/AA simple mixture for analysis. The graph demonstrates that both CS‐R complex and CS‐Q complex exhibit lower TEWL reduction effects immediately after application compared to CS, on par with the effects of glycerin. However, after 1 week, both complexes display significantly enhanced TEWL reduction effects compared to CS solution. This can be attributed to the superior absorbability of CS‐AA complex by the skin, as well as its exceptional water‐holding capacity, resulting in long‐term moisture retention and decreased TEWL. Based on these evaluations, it is anticipated that CS‐AA complex possesses water‐holding capacity both immediately after application and long‐term, through synergistic effects.

FIGURE 4.

Changes of transepidermal water loss (TEWL) after 1 week (comparison between 30 min and 1 week, *p < 0.05).

The effect of CS‐AA complex on increasing water‐holding capacity was confirmed in a previous ex vivo and in vivo study. To determine if these effects are sustained over a longer period, we conducted a 4‐week evaluation of skin hydration (arbitrary units) using the corneometer. Similar to the previous TEWL evaluation, the samples were applied to the forearm of seven participants at the same location, with a concentration of 0.1% based on CS. The skin hydration was analyzed immediately after application and after 4 weeks. As shown in Table 2, the values at week 0, immediately after application, were relatively similar for all cases. However, after 4 weeks, it was observed that the moisture levels increased by 4.4%, 30.65%, and 21.93% when CS, CS‐Q complex, and CS‐R complex were applied, respectively. Although the previous evaluation confirmed improved moisture retention over a short period, these results demonstrate the long‐term improvement of actual skin moisture levels throughout 4 weeks. The results confirm the short‐ and long‐term effectiveness of the CS‐AA complex in improving skin hydration.

TABLE 2.

Changes in skin hydration on forearm after 4 weeks.

	CS	CS‐Q complex	CS‐R complex
0‐week	37.44 ± 8.55	37.65 ± 6.58	39.36 ± 6.60
4‐week	39.09 ± 3.81*	49.19 ± 7.49*	47.99 ± 8.37*

Note: Data are represented as the mean ± SD. Comparison between 0‐ and 4‐week (^* p < 0.05).

Abbreviations: CS, chondroitin sulfate; Q, glutamine; R, arginine.

4. Conclusions

CS‐AA complex, formed through the formation of a polyion complex between CS and AA, demonstrates the potential to be applied as skin hydration ingredients in both short‐ and long‐term applications. The complex effectively inhibits the self‐aggregation of polymers and exhibits a synergistic effect, leading to enhanced moisturizing efficacy. The formation of the CS‐AA complex was confirmed through various analyses, including particle size and zeta‐potential analysis, TEM image analysis, and FT‐IR analysis. Additionally, the complex showed significant improvements in water‐holding capacity, reduction of TEWL, and increased skin hydration. These findings highlight the promising application of the CS‐AA complex in the field of skincare as an effective moisturizing agent.

Ethics Statement

No ethical approval from the committee was needed. All participants in this research were internal company researchers who were fully aware of and actively participated in the experimental procedures and conditions.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data used to support the findings of this study are included within the article or available from the corresponding author upon request.